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Genetic toxicity in vitro

Description of key information

The test substance is not genotoxic under the conditions tested.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Restriction: No strain included that could detect certain oxidising or cross-linking agents (e.g. TA 102); no information on positive control substances
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
The method of Ames, McCann & Yamasaki (1975) was followed.
Deviations:
yes
Remarks:
Deviation from OECD TG 471: No strain included that could detect certain oxidising or cross-linking agents (e.g. TA 102)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: TA92
Details on mammalian cell type (if applicable):
The test strain was originally provided by Dr B. N. Ames, University of California, Berkeley, USA.
Species / strain / cell type:
other: TA94
Details on mammalian cell type (if applicable):
The test strain was originally provided by Dr B. N. Ames, University of California, Berkeley, USA.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The test strains were originally provided by Dr B. N. Ames, University of California, Berkeley, USA.
Metabolic activation:
with and without
Metabolic activation system:
liver microsome fraction (S-9) prepared from the liver of Fischer rats (Charles River Japan Co.)
Test concentrations with justification for top dose:
Six concentrations were tested (max. 5.0 mg/plate) to determine the maximum dose.
Vehicle / solvent:
dimethylsulphoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation with the test sample and with or without the S-9 mix

DURATION Preincubation: 20 min at 37°C

NUMBER OF REPLICATIONS: 2 for each concentration

OTHER: If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays were performed.
Evaluation criteria:
The number of revertant (his+) colonies was scored after incubation at 37°C for 2 days.
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).
Species / strain:
other: TA94
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Species / strain:
other: TA92
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Additional information on results:
The maximum dose was 5.0 mg/plate, which represents the highest non-cytotoxic dose used in the experiment.
Conclusions:
The test item was found not to be genotoxic in an Ames test using the S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98.
Executive summary:

In the current study no OECD guideline was followed and the study was not according to GLP, however, the method of Ames, McCann & Yamasaki (1975) was followed which is similar to the OECD Guideline 471.

The assay included the S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 and the genotoxicity of the test item was investigated with and without metabolic activation, which was the liver microsome fraction (S-9) from Fischer rats.

The test item was found to be negative for all S. typhimurium strains, which indicates that no significant increase in the number of revertant colonies were detected at a maximum dose of 5.0 mg/plate, at which no cytotoxic effects were observed.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
V79 (Chinese hamster) cells at the HPRT locus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30.06.-20.08.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Source: Laboratory for Mutagenicity Testing; Technical University, Germany
- Medium: MEM (minimal essential medium) supplemented with Hank’s salts, 10% Fetal Bovine Serum (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine.
- Culture conditions: 37°C in a 1.5% carbon dioxide atmosphere (98.5% air).
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital / ß-naphthoflavone
Test concentrations with justification for top dose:
The test concentrations were selected based on the pre- experiment.
Experiment I:
4 hours: 6.9; 13.8; 27.5; 55, 110 and 220 µg/mL
Experiment II:
24 hours: 13.8; 27.5; 55, 110 and 165 µg/mL
4 hours: 6.9; 13.8; 27.5; 55, 110 and 220 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium in flask
DURATION: Expression time (cells in growth medium): 7 days
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: stained colonies with more than 50 cells were counted
POSITIVE CONTROLS:
- 4h treatment, without S9 mix: EMS, 150 µg/mL
- 4h treatment, with S9 mix: DMBA, 1.1 µg/mL
- 24h treatment, without S9 mix: EMS, 150 µg/mL
- 24h treatment, with S9 mix: DMBA, 1.1 µg/mL
Evaluation criteria:
The gene mutation assay is considered acceptable if:
a) the amount of mutant colonies per 10e6 cells found in the solvent controls fall within the laboratory historical control data range.
b) the positive control substances produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls exceeds 50%.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Phase separation was noted in experiment I at 110.0 μg/mL and above with and without metabolic activation. In the second experiment phase separation occurred at 220 μg/mL with metabolic activation.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 12.9 up to 28.7 mutants per 10e6 cells; the range of the groups treated with the test item was from 10.4 up to 51.0 mutants per 10e6 cells.
EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

        relative relative relative mutant   relative relative relative mutant  
  conc. P/ S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
  μg/mL PS mix efficiency1 density efficiency II 106 cells factor efficiency1 density efficiency II 106 cells factor
        % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment I / 4 h treatment       culture I culture II
Solvent control     - 100 100 100 22.4 1 100 100 100 15.5 1
Positive control (EMS) 150   - 81 121.9 74.6 231.2 10.3 100.9 116.7 83.1 217.1 14
Test item 6.9   - 101.9 118.4 76.5 25.9 1.2 76.6 101.5 93.8 12.1 0.8
Test item 13.8   - 109.4 86.8 90.2 18.6 0.8 81.7 95.9 100.2 15.8 1
Test item 27.5   - 85.4 91.3 90.3 27.6 1.2 81.7 112 96.5 26.8 1.7
Test item 55   - 91.3 127.3 72.9 49.5 2.2 85.6 130 103.2 37.9 2.5
Test item 110 PS - 56.7 32.3 87.2 24.2 1.1 58.8 32.6 112.6 36.5 2.4
Test item 220 PS - 0 culture was not continued#   culture was not continued#
Solvent control     + 100 100 100 12.9 1 100 100 100 20.1 1
Positive control (DMBA) 2.2   + 79.9 109 87.7 133.2 10.4 78.8 92.3 75.7 267.6 13.3
Test item 6.9   + 100.4 culture was not continued# 94.6 culture was not continued#
Test item 13.8   + 99.1 99.6 88 23.4 1.8 96.7 107.2 94.7 21.9 1.1
Test item 27.5   + 96.9 83.6 86.6 13.2 1 97.4 120 98.3 13 0.6
Test item 55   + 106.6 106.6 82.9 15.8 1.2 94.8 126.3 99.8 44.4 2.2
Test item 110 PS + 98.4 118.1 82.9 28.8 2.2 95.7 123 101.6 16.5 0.8
Test item 220 PS + 101.2 100.6 83.2 19.8 1.5 97.9 111.9 96.1 42.1 2.1
Experiment II / 24 h treatment       culture I culture II
Solvent control     - 100 100 100 28.7 1 100 100 100 19.4 1
Positive control (EMS) 150   - 97.1 81.1 78.2 135.4 4.7 106.5 86.5 93.4 115.9 6
Test item 13.8   - 97.5 72.9 87.6 12.2 0.4 105.6 90.1 96.3 14.9 0.8
Test item 27.5   - 99.4 90.3 82.5 21.8 0.8 100.7 91.7 98.9 11.3 0.6
Test item 55   - 100 104.9 94.3 19.2 0.7 101.5 103.2 88.9 35.3 1.8
Test item 110   - 97 129.9 91.7 22.6 0.8 91.4 101.6 89.9 27 1.4
Test item 165   - 47.9 9.6 78.8 14.8 0.5 51.7 9.4 78.4 51 2.6
Test item 220 PS - 0 1.5 culture was not continued# 0 0 culture was not continued#
Experiment II / 4 h treatment       culture I culture II
Solvent control     + 100 100 100 26.7 1 100 100 100 15.3 1
Positive control (DMBA) 2.2   + 99.6 98.9 91.3 177.5 6.4 92.7 98.7 80.9 194.8 12.7
Test item 6.9   + 92.2 culture was not continued# 91.2 culture was not continued#
Test item 13.8   + 99.4 101.1 98 20.1 0.7 88.3 89.1 87 20.6 1.3
Test item 27.5   + 89.2 100.5 97.6 23.5 0.9 91.8 103.4 90.3 18.1 1.2
Test item 55   + 94.2 91.9 97.3 13.6 0.5 88.6 122 89.1 10.4 0.7
Test item 110   + 82.1 105.7 96.3 22.8 0.8 82.3 106 85.7 12.3 0.8
Test item 220 PS + 75.5 65.5 90.7 17.3 0.6 75.9 92.9 88.8 15 1

PS = Phase separation

# culture was not continued due to exceedingly severe cytotoxic effects

## culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:
Under the experimental conditions here the test item did not induce gene mutations at the HPRT locus in V79 cells, and thus, is considered to be non-mutagenic in this HPRT assay.
Executive summary:

In the current study the potential of the test item to induce gene mutations was assessed at the HPRT locus using V79 cells of the Chinese hamster. The OECD guideline 476 was followed and the study was GLP-compliant.

The V79 cells are exposed to the test item both with and without exogenous metabolic activation. At a defined time interval after treatment the descendants of the treated original population are monitored for the loss of functional HPRT enzyme.

The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The concentration range of the main experiments was limited by cytotoxicity and solubility of the test item.

The tested concentrations were:

Experiment I:

4 hours: 6.9; 13.8; 27.5; 55, 110 and 220 µg/mL

Experiment II:

24 hours: 13.8; 27.5; 55, 110 and 165 µg/mL

4 hours: 6.9; 13.8; 27.5; 55, 110 and 220 µg/mL

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
In vitro micronucleus test in human lymphocytes
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08.07 - 06.08.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
September 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human lymphocytes from healthy non-smoking donors not receiving medication
Details on mammalian cell type (if applicable):
Type and identity of media: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture1:1) supplemented with 200mM GlutaMAXTM, penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and anticoagulant heparin (125 U.S.P.-U/mL).
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital / ß-naphthoflavone
Test concentrations with justification for top dose:
11.4 to 1760.0 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium in culture flasks
DURATION: See 'Any other information'
SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 1000 binucleated cells were evaluated for cytogenetic damage.
DETERMINATION OF CYTOTOXICITY
- Method: CBPI, cytotoxicity is expressed as % cytostasis
POSITIVE CONTROLS:
- Exposure period 4h without S9 mix: MMC, 1.0 µg/mL
- Exposure period 20h without S9 mix: Demecolcin, 125.0 ng/mL
- Exposure period 4h with S9 mix: CPA, 17.5 µg/mL
Evaluation criteria:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).
Statistics:
Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No relevant influence on osmolarity or pH was observed.
Phase separation was observed at the end of treatment at 187.7 μg/mL and above in Experiment I in the absence and presence of S9 mix and at 328.0 μg/mL and above in Experiment II in the absence of S9 mix.
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix and continuous treatment the concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item.
In both experiments, either Demecolcin (125.0 ng/mL), MMC (1.0 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Experiment I

Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs without S9 mix.

Treatment group Conc. Per mL S9 mix Exposure/preparation Cell proliferation culture 1* Proliferation Index CBPI Cell proliferation culture 2* Proliferation Index CBPI CBPI mean Cytostasis [%]
        c1 c2 c4-c8   c1 c2 c4-c8      
Solv. control# 0.50% - 4 / 40 hrs 84 374 42 1.92 100 367 33 1.87 1.89  
Pos. control## 1.0 μg - 4 / 40 hrs 331 164 5 1.35 338 158 4 1.33 1.34 61.8
Test item 61.3 μg - 4 / 40 hrs 76 387 37 1.92 99 361 40 1.88 1.9 n.c.
107.2 μg - 4 / 40 hrs 100 367 33 1.87 75 378 47 1.94 1.91 n.c.
187.7 μg - 4 / 40 hrs 205 288 7 1.6 150 342 8 1.72 1.66 25.9

* c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

# DMSO

## MMC

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs with S9 mix

Treatment group Conc. Per mL S9 mix Exposure/preparation Cell proliferation culture 1* Proliferation Index CBPI Cell proliferation culture 2* Proliferation Index CBPI CBPI mean Cytostasis [%]
        c1 c2 c4-c8   c1 c2 c4-c8      
Solv. control# 0.50% + 4 / 40 hrs 80 381 39 1.92 126 348 26 1.8 1.86  
Pos. control## 17.5 μg + 4 / 40 hrs 277 215 8 1.46 256 235 9 1.51 1.48 43.7
Test item 61.3 μg + 4 / 40 hrs 96 368 36 1.88 100 365 35 1.87 1.88 n.c.
107.2 μg + 4 / 40 hrs 131 338 31 1.8 158 314 28 1.74 1.77 10.4
187.7 μg + 4 / 40 hrs 129 342 29 1.8 132 339 29 1.79 1.8

7.2

* c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

# DMSO

## MMC

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

Number of micronucleated cells; exposure period 4 hrs without S9 mix

Treatment group Conc. per mL S9 mix Exposure/preparation Binucleate cells with n micronuclei culture 1 sum culture 1 Micronucleated cells Binucleate cells with n micronuclei culture 2 sum culture 2 sum in 2000 binucleate cells  [%]
        1 2 >2   1 2 >2      
Solv. control# 0.50% - 4 / 40 hrs 4 0 0 4 5 0 1 6 10 0.5
Pos. control## 1.0 μg - 4 / 40 hrs 153 19 2 174 179 11 5 195 369 18.45
Test item 61.3 μg - 4 / 40 hrs 4 1 0 5 9 0 0 9 14 0.7
107.2 μg - 4 / 40 hrs 5 0 0 5 7 0 1 8 13 0.65
187.7 μg - 4 / 40 hrs 5 0 0 5 6 1 0 7 12

0.6

# DMSO

## MMC

Number of micronucleated cells; exposure period 4 hrs with S9 mix

Treatment group Conc. per mL S9 mix Exposure/preparation Binucleate cells with n micronuclei culture 1 sum culture 1 Micronucleated cells Binucleate cells with n micronuclei culture 2 sum culture 2 sum in 2000 binucleate cells  [%]
        1 2 >2   1 2 >2      
Solv. control# 0.50% + 4 / 40 hrs 5 0 0 5 4 1 0 5 10 0.5
Pos. control## 17.5 μg + 4 / 40 hrs 57 4 0 61 60 1 0 61 122 6.1
Test item 61.3 μg + 4 / 40 hrs 7 0 0 7 5 0 0 5 12 0.6
107.2 μg + 4 / 40 hrs 4 1 0 5 3 0 0 3 8 0.4
187.7 μg + 4 / 40 hrs 4 0 0 4 7 0 0 7 11 0.55

# DMSO

## MMC

Experiment II

Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 20 hrs without S9 mix

Treatment group Conc. Per mL S9 mix Exposure/preparation Cell proliferation culture 1* Proliferation Index CBPI Cell proliferation culture 2* Proliferation Index CBPI CBPI mean Cytostasis [%]
        c1 c2 c4-c8   c1 c2 c4-c8      
Solv. control# 0.50% - 20 / 40 hrs 40 400 60 2.04 69 381 50 1.96 2  
Pos. control## 125.0 ng - 20 / 40 hrs 123 367 10 1.77 124 358 18 1.79 1.78 22
Test item 35.0 μg - 20 / 40 hrs 29 421 50 2.04 47 417 36 1.98 2.01 n.c.
61.3 μg - 20 / 40 hrs 45 417 38 1.99 65 404 31 1.93 1.96 4.2
107.2 μg - 20 / 40 hrs 99 385 16 1.83 73 411 16 1.89 1.86 14.1
187.7 μg - 20 / 40 hrs 274 222 4 1.46 352 144 4 1.3 1.38 61.8

* c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

# DMSO

## Demecolcin

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

Number of micronucleated cells; exposure period 20 hrs without S9 mix

Treatment group Conc. per mL S9 mix Exposure/preparation Binucleate cells with n micronuclei culture 1 sum culture 1 Micronucleated cells Binucleate cells with n micronuclei culture 2 sum culture 2 sum in 2000 binucleate cells  [%]
        1 2 >2   1 2 >2      
Solv. control# 0.50% - 20 / 40 hrs 6 0 0 6 4 1 0 5 11 0.55
Pos. control## 125.0 ng - 20 / 40 hrs 22 5 2 29 28 6 0 34 63 3.15
Test item 35.0 μg - 20 / 40 hrs 6 0 0 6 4 0 0 4 10 0.5
61.3 μg - 20 / 40 hrs 10 0 0 10 8 1 0 9 19 0.95
107.2 μg - 20 / 40 hrs 3 0 0 3 4 0 0 4 7 0.35

# DMSO

## Demecolcin

Conclusions:
The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and is therefore considered to be not mutagenic.
Executive summary:

In the current study the potential of the test item to induce chromosomal aberrations was investigated in an in vitro micronucleus assay. The OECD guideline 487 was followed and the study was GLP-compliant.

The induction of cytogenetic damage in human lymphocytes (from healthy non-smoking donors) was assessed in two independent experiments.

Cytochalasin B was used as cytokinesis blocker to ensure that only binucleate cells were evaluated for micronuclei.

Treatments started after a 48 hour stimulation period with phytohemeagglutinine (PHA) when cells were actively proliferating and the cells were prepared at approximately 2 – 2.5 fold of the normal cell cycle time.

For validation of the test, control mutagens were tested in parallel to the test item.

In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (1760.0 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment II in the absence of S9 mix and continuous treatment, the concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Therefore, under the experimental conditions reported here, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and is considered to be not mutagenic (not clastogenic and not aneugenic).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For the assessment of genetic toxicity there are three key studies (in vitro tests) available: one Ames test, one in vitro micronucleus test and one in vitro gene mutation test in mammalian cells (HPRT). As supporting studies two additional tests are available: one sister chromatid exchange assay and one chromosomal aberration test.

 

In the first study no OECD guideline was followed, however, the method of Ames, McCann & Yamasaki (1975) is similar to the OECD Guideline 471. S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 were included in the study and the genotoxicity was investigated with and without metabolic activation (S9). The test item was negative in all strains, no significant increase in revertant colonies was detected up to the maximum dose of 5.0 mg/plate.

 

In the second study the potential of the test item to induce chromosomal aberrations was investigated in an in vitro micronucleus assay according to OECD 487. The induction of cytogenetic damage in human lymphocytes was assessed in two independent experiments.

In each experimental group two parallel cultures were analysed for cytogenetic damage. The highest concentration was 1760.0μg/mL.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Therefore, under the experimental conditions reported here, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and is considered to be not mutagenic (not clastogenic and not aneugenic).

 

In the third study the potential of the test item to induce gene mutations was assessed in Chinese hamster cells according to OECD 476. The V79 cells were exposed to the test item both with and without exogenous metabolic activation and after treatment the descendants of the treated cells were monitored for the loss of functional HPRT enzyme. The cells were exposed for 4 hours in the first experiment with and without metabolic activation, and in the second experiment for 4 and 24 hours with and without metabolic activation.

The concentration range of the main experiments was limited by cytotoxicity and solubility of the test item.

The tested concentrations were in Experiment I: 6.9; 13.8; 27.5; 55, 110 and 220 µg/mL and in Experiment II: for 24 hours: 13.8; 27.5; 55, 110, 165 and 220 µg/mL; for 4 hours: 6.9; 13.8; 27.5; 55, 110 and 220 µg/mL.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments while the positive controls induced a distinct increase in mutant colonies. Under the experimental conditions the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

 

In the first supporting study the potential of the test item to modify MMC-induced sister-chromatid exchanges (SCEs) was assessed. No OECD guideline was followed, however, the method is comparable to OECD 479. Cultured Chinese hamster CHO K-1 cells were used. No effect on the cell cycle and spontaneous SCEs were observed. However, an increase in SCE frequency occurred by post-treatment with the test substance on MMC-treated cells.

 

In the second supporting study the potential of the test item to induce chromosome aberrations was assessed in a study similar to the OECD 473. Chinese hamster fibroblast cells were exposed for 24 and 48 hours, without metabolic activation. Chromosome preparations were made and 100 well-spread metaphases were observed under the microscope. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, were recorded.

The test item was found to be equivocal because even though polyploidisation effects were observed, the percentage was below 10% and no structural aberrations were observed after 48 hours. The maximum dose of the test item was 0.063 mg/mL.

 

In conclusion, the test item did not induce gene mutations or chromosome aberrations in 5 independent studies. Therefore, the test item is considered to be non-mutagenic.

Justification for classification or non-classification

The test item did not induce gene mutations or chromosome aberrations in 5 independent studies.

According to the criteria set in the Regulation EC No 1272/2008 in section 3.5 the test item is not to be considered as genotoxic/mutagenic.

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation EC No 1272/2008, and are therefore conclusive but not sufficient for classification.